An improved Agrobacterium mediated transformation in tomato using hygromycin as a selective agent

Abstract

Bacterial wilt is a devastating disease of tomato crop throughout the world. This disease is very dangerous in hot and humid regions, where it spreads with the irrigation water to whole field within days, which resulted in severe decline in yield. Two varieties of tomato were used for developing bacterial wilt resistance. Riogrande responded more efficiently as compared to Roma. Regeneration frequency of 90.6% was achieved when leaf discs were used and 82.5% when hypocotyls were used as explant for tomato cv. Riogrande. While 65.4% regeneration was achieved from hypocotyls and 72.6% regeneration was obtained when leaf discs were used as explant for cv. Roma. Explants were cocultivated with Agrobacterium strain EHA101 containing a binary vector pTCL5, having hygromycin phosphotransferase (HPT) gene which confers resistance to hygromycin and β-glucuronidase (GUS) gene in addition to Xa21 gene. Hygromycin (25 mg/l) was used as selectable marker while GUS is a reporter gene. Acetosyringone (50 M) enhanced transformation effeciency. Preselection period of 7 days was found to be indispensable for successful transformation of tomato crop. Transformation efficiency of 24% was observed for Riogrande and 8% for Roma. Molecular analysis of transgenic plants produced was carried out for hygromycin resistance gene. Transgenic plants contained the expected band of 670 bp. PCR analysis confirmed the foreign DNA in to the plant genome.